This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

We assessed the expression of cytokeratin (CK) and apomucin (MUC) in ampullary carcinoma
(AC) to develop a system for the classification of ACs on the basis of their clinical
significance.

Method

We studied the expressions of CK7, CK20, MUC1, MUC2, MUC5AC, and MUC6 in 43 patients
with ACs. Clinical data were obtained retrospectively by examining surgically resected
ACs of the patients.

Results

We classified the cases into 3 groups: tumors expressing CK20 and lacking MUC1 (intestinal
type [I-type], 26%), tumors expressing MUC1 and lacking CK20 (pancreatobiliary type
[PB-type], 35%), and those expressing or lacking both CK20 and MUC1 (other type [O-type],
39%). Eight (73%) of 11 I-type carcinomas, 3 (20%) of 15 PB-type carcinomas, and 4
(24%) of 17 O-type carcinomas were classified as pT1. The number of I-type carcinomas
in the early tumor stages was significantly higher than the number of PB- and O-type
carcinomas (p = 0.014 and p = 0.018, respectively). The 5-year survival rates for
pT1, pT2, and pT3 tumors were 76%, 33%, and 22%, respectively (p < 0.001). Rates of
MUC5AC and MUC6 coexpression for I-type, PB-type, and O-type tumors were 18%, 13%,
and 53%, respectively. There was a significant correlation between MUC5AC and MUC6
coexpression and O-type characteristics (p = 0.031). The five-year survival rates
for O-type ACs with and without MUC5AC and MUC6 coexpression were 71% and 17%, respectively
(p = 0.048).

Conclusions

The immunohistochemical subtypes based on CK and MUC expression correlated with tumor
progression. Gastric MUC5AC and MUC6 coexpression correlated with better prognosis
for O-type ACs.

Background

Ampullary carcinomas (ACs), although uncommon, have a better prognosis than other
periampullary tumors such as pancreatic and bile duct carcinomas.

The ampulla of Vater consists of 4 minor anatomic regions: the ampulloduodenum (Ad),
the ampullopancreatobiliary common duct (Ac), the ampullopancreatic duct (Ap), and
the ampullobiliary duct (Ab)[1,2]. The ampulla is formed by the union of 2 distinct types of mucosa. The Ad is covered
by intestinal mucosa, while the other parts of the ampulla of Vater (the Ap, Ab, and
Ac) are lined with pancreatobiliary-type ductal mucosa[2,3]. Therefore, ACs may arise from the intestinal-type mucosa as well as from the pancreatobiliary-type
mucosa; this may explain the broad histomorphologic spectrum of these tumors[1]. Tumor progression and prognosis are affected by the primary AC tumor sites [1,3,4].

Kimura et al. classified ACs into 2 histological subtypes: intestinal and pancreatobiliary[3]. Albores-Saavedra et al. further defined the characteristics of these 2 types and
also described unusual types such as signet-ring cell carcinoma and undifferentiated
carcinoma[4].

While histopathological typing is a useful method for classifying ACs, some cases
cannot be easily classified by using histomorphology[1]. Determination of the cytokeratin (CK) and apomucin (MUC) immunophenotypes of an
AC can facilitate identification of the primary tumor site[1,2,5-7]. Most pancreatobiliary adenocarcinomas express CK7 and low levels of CK20[6,8,9]. Among ACs, the pancreatobiliary type expresses CK7 but does not express CK20, while
the intestinal type expresses CK20 but does not express CK7[5].

The pancreatobiliary type of ACs usually express MUC1 but do not express MUC2[6,7,9,10]. Most intestinal-type ACs express MUC2[1,2,5,6].

In the present study, we analyzed the spectrum of CK and MUC expression in 43 patients
with ACs. We then evaluated the immunohistochemical subtypes of ACs by analyzing the
expressions of CK7, CK20, MUC1, MUC2, MUC5AC, and MUC6 in these tumors. Further, we
assessed the correlations between the histomorphological findings and the defined
immunohistochemical subtypes and evaluated the clinical significance of these immunohistochemical
AC subtypes; the classification of ACs on the basis of their immunohistochemical characteristics
may be useful to predict the clinical outcome.

Materials and methods

Clinical data were obtained retrospectively from ACs that were surgically resected
from 43 patients (22 men and 21 women) with an average age of 66.4 years (range, 44-82
years). All resected specimens had been obtained between 1983 and 2007 and were maintained
at the Department of Digestive and General Surgery, Faculty of Medicine, Shimane University.
All but 5 patients underwent pancreatoduodenectomy. The other 5 underwent pancreas-sparing
duodenectomy. All tumors included in this study histologically showed surgically negative
margins.

The study was approved by the hospital's ethics committee. Informed consent was obtained
from all patients for the subsequent use of resected tissues.

Histopathological examinations were performed according to the guidelines of the Japanese
Society of Biliary Surgery[11]. The Tumor, Node, Metastasis (TNM) Staging System put forth by the International
Union Against Cancer was used for tumor classification[12]. All tumors were classified histologically according to the criteria published by
Albores-Saavedra et al[4]. Intestinal-type carcinomas are composed of well-formed tubular to elongated glands,
complex cribriform areas, and solid nests indistinguishable from those found in colorectal
adenocarcinoma, whereas pancreatobiliary-type carcinomas mostly consist of simple
or branching glands and small solid nests of cells surrounded by a strikingly desmoplastic
stroma (Figure 1). Mixed-pattern tumors were classified into the intestinal- or pancreatobiliary-type
groups on the basis of their predominant component. Carcinomas of the unusual types
included undifferentiated, mucinous, signet-ring cell, and solid carcinomas.

Immunohistochemical staining was performed on an immunostainer (Benchmark XT; Ventana
Medical System, Tucson, AZ) with the use of an amplification kit (Ventana). Antibody
detection was performed by adding biotinylated secondary antibodies, avidin-biotin
complex, and 3,3'-diaminobenzidine.

The cytoplasmic and membranous immunoreactivities of CK7, CK20, MUC1, MUC2, MUC5AC,
and MUC6 were assessed. Only those samples showing greater than 10% tumor-cell positivity
were regarded as positive.

Statistical Analysis

Survival curves were calculated by the Kaplan-Meier method and compared with the results
of the log-rank test. Agreement between the histological and immunohistochemical classifications
was evaluated using the κ-coefficient. A two-tailed Fisher's exact test or χ2 test was used to compare the immunohistochemical classification and clinicopathological
parameters, as appropriate. Probability (p) values of < 0.05 obtained by the two-tailed
test were regarded as statistically significant.

The JMP software program (ver. 5.0.1; SAS Institute Inc, Cary, NC) was used for all
statistical analyses.

Results

Patient Characteristics and Histological Classification

Fifteen (35%) of the 43 tumors were pT1, 11 (25%) were pT2, and 17 (40%) were pT3.
Twenty-two (51%) and 21 (49%) of the 43 tumors were negative and positive for lymph
node involvement, respectively.

In the assessment according to the histological criteria proposed by Albores-Saavedra
et al.,[4] we found 16 (37%) tumors to be intestinal-type carcinomas, 18 (42%) to be pancreatobiliary-type
carcinomas, and 9 (21%) to be unusual-type carcinomas (Table 1).

The histological classification[4] indicated that CK20 had high sensitivity (100%) for intestinal-type carcinoma and
that MUC1 had high sensitivity (94%) for pancreatobiliary-type carcinoma (Table 2, Figure 1), and both correlations were significant (p < 0.001 and p < 0.001, respectively).

Immunohistochemical Classification of Tumors

We tried to further classify ACs into 3 subtypes on the basis of the expression of
CK20 and MUC1 in the intestinal mucosa and the pancreatobiliary mucosa: tumors expressing
CK20 and lacking MUC1 were defined as intestinal type (I-type); tumors expressing
MUC1 and lacking CK20 were defined as pancreatobiliary type (PB-type); and carcinomas
expressing or lacking both CK20 and MUC1 were defined as other type (O-type). Eleven
(26%) of the 43 tumors were I-type, 15 (35%) were PB-type, and 17 (39%) were O-type.

Ten of the 16 carcinomas of the histological intestinal type were of the immunohistochemical
I-type, but none was of the immunohistochemical PB-type. Thirteen of the 18 carcinomas
of the histological pancreatobiliary type were of the immunohistochemical PB-type,
while none was of the immunohistochemical I-type (Table 3). The sensitivity of the marker for the coexpression of CK20 and MUC1 for the histological
intestinal type, pancreatobiliary type, and unusual type of tumors were 63%, 72%,
and 67%, respectively, and the specificity for these types were 96%, 92%, and 68%,
respectively (Table 4). Further, the immunohistochemical subtypes defined in our study correlated well
with the conventional histological classification (κ-coefficient = 0.518; p < 0.001).

Survival Analysis

The survival time of the 43 patients was 4.23 ± 0.64 years (mean ± SEM, Kaplan-Meier).
The 5-year survival rate for all AC cases was 41.2%. The 5-year survival rates for
pT1, pT2, and pT3 cases were 76%, 33%, and 22%, respectively (Figure 2). The 5-year survival rates for node-negative and node-positive cases were 61% and
19%, respectively (Figure 3). There was a significant difference in the cumulative survival between pT and N
cases (p < 0.001 and p < 0.001, respectively). The 5-year survival rates for the histological
intestinal, pancreatobiliary, and unusual types were 62%, 33%, and 25%, respectively
(p = 0.034) (Figure 4). However, the 5-year survival rates for the immunohistochemical I-type, PB-type,
and O-type were 55%, 35%, and 41%, respectively; therefore, we concluded that there
was no significant correlation between survival and immunohistochemical subtypes (p
= 0.560) (Figure 5).

Figure 4.Cumulative survival of patients with ACs of different histological types: intestinal-type,
pancreatobiliary-type, and unusual-type. Patients with intestinal-type tumors showed significantly better survival than those
with the pancreatobiliary- and unusual-type tumors (p = 0.031).

Figure 5.Cumulative survival of patients based on immunohistochemical classification (I-type,
PB-type, and O-type). There were no differences among the 3 immunohistochemical subtypes in terms of cumulative
survival of patients with AC (p = 0.56).

Relationships between MUC5AC and MUC6 Expression and Immunohistochemical Subtypes

The rate of coexpression of MUC5AC and MUC6 in tumors of the immunohistochemical I-type,
PB-type, and O-type were 18%, 13%, and 53%, respectively. Significant correlations
were noted between MUC5AC and MUC6 coexpression and immunohistochemical subtypes (p
= 0.031) (Figure 6) (Table 7). For the immunohistochemical O-type, the 5-year survival rates of patients with
tumors coexpressing MUC5AC and MUC6 and with those lacking both antigens were 71%
and 17%, respectively.

Among the patients with immunohistochemical O-type tumors, those with tumors coexpressing
MUC5AC and MUC6 had significantly longer cumulative survival than those with tumors
that did not show this coexpression (p = 0.048) (Figure 7).

Figure 7.Cumulative survival of patients with immunohistochemical other-type tumors. Patients with tumors showing coexpression of MUC5AC and MUC6 showed significantly
better survival than those with tumors negative for expression of both MUC5AC and
MUC6 (p = 0.048).

Discussion

In the present study on ACs, we histologically classified these tumors into these
3 types, and 37% were of the intestinal type, 42% were of the pancreatobiliary type,
and 21% were of the unusual type[4]. According to the histological criteria, Zhou et al. reported the rates of the histological
intestinal, pancreatobiliary, and unusual types to be 27%, 44%, and 29%, respectively[5]. According to their histological criteria, the unusual type included mucinous, signet-ring
cell, solid, or undifferentiated carcinomas[4,5]. However, Kimura et al. simply classified ACs into 2 types, intestinal and pancreatobiliary[3].

Because these systems of histological classification use different sets of criteria,[3,4] we tried to create a simple AC classification system based on immunohistochemical
staining of CKs and MUCs. Goldstein et al. reported positive CK7 expression in 100%
and positive CK20 expression in 43% of ACs, but it was difficult to distinguish between
pancreatic carcinomas and ACs by the coordinate staining patterns of CK7 and CK20[9]. Very few reports have examined the expression patterns of CK and MUC in the AC subtypes[1,5,6].

In a previous study, the CK7+/CK20-/MUC2- pattern in the histological intestinal-type
carcinoma and the CK7-/CK20+/MUC2+ pattern in the histological pancreatobiliary-type
carcinoma indicated that these different types of ACs had developed from 2 different
types of mucosa in the ampulla of Vater[1]. Chu et al. reported positive expression of CK7, CK20, and MUC2 in the histological
intestinal-type and positive expression of CK7 and MUC1 in the histological pancreatobiliary-type
and that ACs of pancreatobiliary origin showed immunophenotypes similar to that of
pancreatic ductal carcinoma[6]. Zhou et al. were the first to show agreement between the histological classification
and the immunohistochemical characterization based on cytokeratins[5]. However, their immunohistochemical classification did not correlate with tumor progression
and prognosis.

Although most other studies have described immunohistochemical classification systems
based on the expression of either MUC[2] or CK[5], we analyzed the expression of both in ACs.

In the present study, significant differences were noted in the expression levels
of the histological intestinal type and the histological pancreatobiliary type, with
the sensitivity being 100% for CK20 and 94% for MUC1 expression, respectively. These
results indicate that the CK20+/MUC1- pattern fully corresponds to the immunohistochemical
I-type and that the CK20-/MUC1+ pattern fully corresponds to the immunohistochemical
PB-type.

With regard to immunohistochemical classification systems, Zhou et al classified ACs
on the basis of the combined expression of CK7 and CK20, while Chu et al classified
ACs on the basis of the combined expression of CDX2, CK17, MUC1, and MUC2.

Little is known, however, about the combined expression of CK20 and MUC1 in ACs. The
possibility of identifying the primary AC site is increased when the combined expression
of CK and MUC, rather the expression of either one of them, is taken into account.

We found that the classification of the immunohistochemical subtypes based on the
expression of both CK20 and MUC1 correlated well with histological typing (κ-coefficient
= 0.5184). Using this immunohistochemical classification system based on the coordinated
expression of CK20 and MUC1, we were able to determined that 2 of 9 tumors classified
as the histological unusual type expressed the pancreatobiliary pattern in immunohistochemical
analysis, while 1 of these 9 tumors expressed the intestinal pattern.

Previous studies have shown that the prognosis of AC patients depends on the pT stage,
nodal metastasis, and histological type[1,4,13-17]. Similarly, our results indicated that the pT stage, nodal metastasis, and histological
subtype correlated significantly with cumulative survival. According to our histological
classification method, a large number of intestinal type ACs were at stage pT1 (60%)
and node-negative (81%). These results indicate that progression of the intestinal-type
tumor is slower and the risks of nodal metastasis lower than those in the case of
the pancreatobiliary- and unusual-type tumors.

In our immunohistochemical classification, the pT stage correlated significantly with
the immunohistochemical subtypes. Although the number of immunohistochemical I-type
tumors in the early pT stages was significantly greater than the number of immunohistochemical
PB-type and O-type tumors in the same stages, there were no significant differences
in the cumulative survival. This result may be attributable to the lack of differences
in the nodal metastasis risks associated with the immunohistochemical I-type and PB-type.
This may indicate that the risks of nodal metastasis are similar among the intestinal,
pancreatobiliary, and other types. Therefore, pancreatoduodenectomy with lymph node
dissection should be performed for adequate surgical resection in AC[18]. If function-preserving surgery has been selected for AC, the histological classification
system is currently more useful than our immunohistochemical classification method.

Gürbüz et al. reported that MUC5AC- and MUC6-positive expression patterns were regarded
as representing gastric differentiation and that negative expression of both indicated
the intestinal type of AC[7]. Zhou et al. showed that gastric MUC5AC expression correlated well with PB-type carcinomas[5]. Interestingly, we found that the frequency of the coexpression of gastric MUC5AC
and MUC6 in the immunohistochemical O-type was significantly higher than those in
the immunohistochemical I-type or PB-type. These results provide evidence that both
the immunohistochemical I-type and immunohistochemical PB-type have low-grade expression
of gastric mucins. Expression of these gastric MUCs in the immunohistochemical O-type
indicates that both gastric foveolar and pyloric gland metaplasia occur in the immunohistochemical
O-type[19-21]. In addition, patients with tumors of the immunohistochemical O-type and MUC5AC and
MUC6 coexpression had a significantly longer survival than those with tumors that
did not show this coexpression. Among the tumors of the immunohistochemical O-type,
those negative for the coexpression were at a more advanced pT stage than those positive
for the coexpression. The prognosis of tumors of the immunohistochemical O-type that
were positive or negative for the coexpression may depend on the tumor stage. Thus,
gastric differentiation of the immunohistochemical O-type is associated with good
prognosis. Interestingly, our observations of the histological unusual type were similar.

In summary, we studied the expression of CK7, CK20, MUC1, MUC2, MUC5AC, and MUC6 in
ACs. On the basis of the histological classification of ACs, we found that CK20 had
a high sensitivity for the histological intestinal type and MUC1 had high sensitivity
for the histological pancreatobiliary type carcinoma. On the basis of the observed
differences in the expression patterns of both CK and MUC, we defined immunohistochemical
subtypes. These immunohistochemical subtypes correlated well with the conventional
histomorphological classification but did not correlate with prognosis. However, the
coexpression of gastric MUC5AC and MUC6 correlates with the prognosis of patients
with the immunohistochemical O-type of AC.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

YK participated in the design of the study, histological diagnoses, data processing,
and drafting of the manuscript. TT participated in the design of the study and reviewed
the manuscript. TN established histological diagnoses and reviewed the manuscript.
TI, TN, and SY participated in the design of the study, established histological diagnoses,
and reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The expert technical assistance of Ms Risa Satou and Mrs. Miki Asazu with the immunohistochemistry
is highly appreciated.